1. Field of the Invention
The present invention relates to methods of forming a humidity barrier using fats or other materials that can form an amorphous coating for particles comprising sensitive biological materials in a dehydrated and/or a vitrified state. Since the hydrophobic coating may be sticky, a second hard-shell coating is applied to prevent the particles from sticking together. This approach would solve a significant problem in achieving long-term shelf preservation of sensitive biological materials by minimizing the uncontrolled exposure to moisture and the subsequent loss of biological activity.
2. Description of the Related Art
The field of biological preservation encompasses a wide variety of methods used to preserve biological activity in sensitive materials. These preservation methods include refrigeration, freezing, freeze-drying (lyophilization), spray-drying, and more recently, preservation by foam formation (U.S. Pat. No. 5,766,520, incorporated herein by reference). The field of probiotics, which seeks to use the beneficial biological properties of certain types of bacterial strains to promote health in both humans and animals via nutritional and food supplements, presents a particular need for effective ambient temperature preservation, that is not met by present methods. Preservation of probiotic strains can be accomplished using any of the above methods. Moreover, ambient and even elevated temperature preservation of bacterial has been accomplished by dehydration through foam formation or spray drying.
However, none of these methods provide effective protection against the combination of humidity and ambient temperatures, both of which may be encountered by venders as well as consumers, unless special packaging materials or storage environments are utilized. For example, a low humidity storage container, possibly with added desiccants such as cotton or silica, may be used for packaging a pharmaceutical composition such as aspirin. In addition, humidity barrier packaging materials such as aluminum foil may be used for lyophilized or foam-preserved products. Glass containers may be used for frozen, lyophilized, foam-preserved and refrigerated products. In each case the protection provided by the packaging or storage environment is compromised as soon as the container is opened to the atmosphere, thereby exposing atmospheric moisture to the product.
The rate of moisture ingress and the resulting damage to the biological activity of the material is dependent on the nature of the preserved material, the temperature, and the physical state of the material. For dried products, exposure to moisture may cause either rapid (i.e. minutes) or slower (days to months) deterioration of biological activity, depending on the relative degree of protection offered by the method of preservation used. For refrigerated or frozen and thawed products, biologically active components, such as active cultures in yogurt, will have a limited shelf-life from the time it is placed into the package, typically measured in days.
Accordingly, a need remains for methods that combine the preservation of sensitive biological materials by drying with the protection of these dried materials from the deleterious effects of environmental moisture, wherein the biological activity of the dried materials are retained over a broad range of storage temperatures and humidities. Furthermore, the need exists for a humidity barrier that is not compromised by exposure of the preserved product to the atmosphere.
The present invention relates to a method for providing long-term ambient temperature preservation of a sensitive biological sample, having a biological activity. The method includes preserving the sensitive biological sample by drying under conditions wherein at least a portion of the biological activity is retained. The dried sample is milled to form a particulate preparation. The particulate preparation is coated with a first layer comprising a hydrophobic substance. A second layer is applied over the first layer, the second layer forming a hard shell at ambient temperature.
In one aspect of the present invention, the sensitive biological sample may be selected from the group consisting of a bacterium, a pharmaceutical composition, a vaccine, and a nutritional supplement.
The hydrophobic substance in accordance with a preferred method is a fat or an oil. Preferably, the fat or oil is selected from the group consisting of cotton-seed, corn, palm, soy, grapeseed (canola), cod-liver and other fish-based oils, omega-3 fatty acids, neem, olive, peanut, poppy, safflower, sesame, wheat-germ, and the like.
Coating of the particulate preparation with the second layer may be accomplished using a device adapted to produce a high shear mixing action. The second layer preferably comprises a material selected from the group consisting of a sugar, a protein, a polymer, or a mixture thereof.
In one preferred embodiment of the present method, the sensitive biological sample may be preserved by drying the sample in the presence of a protectant formulation. The sensitive biological sample and protectant formulation may be boiled under vacuum to form a mechanically stable foam.
In addition, or in the alternative, the sensitive biological sample may be preserved by drying the sample under a vacuum at a temperature greater than 20xc2x0 C. for a period of time sufficient to increase the glass transition temperature of the sample above 20xc2x0 C.
The present invention also relates to a storage particle adapted for long-term ambient temperature preservation of a sensitive biological material. The storage particle comprises a dehydrated preparation of the sensitive biological material, a first layer comprising a hydrophobic substance which surrounds the dehydrated preparation of the biological material, and a second layer comprising a hard shell at ambient temperature which surrounds the first layer. The first layer minimizes exposure of the dehydrated preparation of the biological material to atmospheric moisture.